Increased recovery of active proteins

ABSTRACT

The invention provides methods of increasing yields of desired conformation of proteins. In particular embodiments, the invention includes contacting preparations of a recombinant protein with a reduction/oxidation coupling reagent for a time sufficient to increase the relative proportion of a desired configurational isomer.

[0001] This application claims the benefit of provisional U.S.application No. 60/271,033, filed Feb. 23, 2001, the disclosure of whichis incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention is in the field of treatment and purification ofproteins.

BACKGROUND

[0003] High levels of expression of many proteins of eukaryotic originhave been achieved in prokaryotic expression hosts. Such eukaryoticproteins often misfold and accumulate as insoluble inclusion bodies inthe prokaryotic host. In order to obtain biologically active protein,the proteins trapped in inclusion bodies had to be unfolded and refoldedunder harsh conditions including chaotropic agents and reducing thiols.

[0004] Expression of proteins of eukaryotic origin in eukaryotic hostsavoided these problems. Provided that the expression vector was properlydesigned (e.g., with secretory signal peptides, etc.), eukaryotic celllines tend to correctly process and secrete extracellular eukaryoticproteins as soluble products.

[0005] However, as expression systems and vectors have been improved tomaximize levels of expression from eukaryotic hosts, not all of therecombinant protein expressed and secreted from these hosts is in thedesired, most active conformation. The invention is designed to overcomesuch expression problems, and maximize yields of biologically activeprotein.

SUMMARY OF THE INVENTION

[0006] The invention is based, in part, on the discovery that not all ofthe preparation of recombinant protein that is expressed by eukaryotichost cells is folded into a native tertiary conformation. In addition,it has been found that regions or domains of recombinant proteins may beproperly folded, while other regions or domains may have undesiredconformations. Accordingly, in one aspect, the invention provides amethod of contacting a preparation of the recombinant protein thatcontains a mixture of at least two isomers of the recombinant protein toa reduction/oxidation coupling reagent for a time sufficient to increasethe relative proportion of the desired conformational isomer anddetermining the relative proportion of the desired conformational isomerin the mixture. In another aspect, the invention entails contacting apreparation of a recombinant protein that has been produced by mammaliancells with a reduction/oxidation coupling reagent, at a pH of about 7 toabout 11, and isolating a fraction of the preparation of the recombinantprotein with a desired conformation. Preferred recombinant proteins areglycosylated recombinant proteins such as, e.g., those produced byeukaryotic cells. The invention also relates to methods of formulatingthe resulting preparations into a sterile unit dose form, andcompositions produced by the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1. Hydrophobic interaction chromatography (HIC) of TNFR:Fc.This preparation of TNFR:Fc elutes during HIC as three distinct peakscollected into Fraction #2 and Fraction #3, as indicated.

[0008]FIG. 2. Circular Dichroism Analysis of Fractions #2 and #3.Near-UV Circular Dichroism measurements expressed in terms of meanresidue ellipticity are shown in FIG. 2. FIG. 2A presents the spectraldata; The line for Fraction #3 is closest to the arrow highlighting thenegative displacement at about 270 nM ascribed to disulfidecontributions, and the line for Fraction #2 is the darker solid line.FIG. 2B presents the curve-fitted data for Fraction #2 (small dashedline) and Fraction #3 (larger dashed line).

[0009]FIG. 3. Molecular Weight Determination Using On-line sizeexclusion chromatography (SEC), ultraviolet (UV), light scattering (LS),and refractive index (RI) detection in series (On-line SEC/UV/LS/RI).FIG. 3A is Fraction #3, and FIG. 3B is Fraction #2. Vertical dashedlines indicate where the slices were evaluated for molecular weightdetermination in the region surrounding the main peak.

[0010]FIG. 4. Differential Scanning Calorimetry Analysis of Fractions #2and #3. FIG. 4A is the uncorrected data, and FIG. 4B presents thebaseline-corrected data. Thermal melting transitions are labeled byvertical dashed lines. Arrows indicate an enthalpy displacement. Thehorizontal dotted lines in FIG. 4B are used as a baseline reference.

[0011]FIG. 5. Correlation of Fraction #2 and Binding Activity. Sixdifferent preparations of TNFR:Fc (denoted A through F), from sixdifferent cell lines, were tested for the correlation between thepercent increase in proportion of Fraction #2 (dark diamonds) andpercent increase in TNF alpha Binding Units (light diamonds).

[0012]FIG. 6. Effect of Varying Cysteine Concentration on Conversion ofFraction #3 into Fraction #2. Protein samples were treated with variousconcentrations of cysteine (0.25-5.0 mM) and changes in Fraction #3assessed using HIC. Four different lots of TNFR:Fc were treated for 18hours at the indicated cysteine concentration on the x-axis. The percentof Fraction #3 in each lot that was converted into Fraction #2 isplotted on the y-axis.

[0013]FIG. 7. Effect of Cysteine Concentration on Proportion of Fraction#3. Protein samples from four different lots were treated with variousconcentrations of cysteine (0-50 mM) and the resulting level of Fraction#3 was assessed by HIC.

[0014]FIG. 8. Effect of Temperature on Disulfide Exchange. Proteinfractions were treated at room temperature or 4 degrees C. in thepresence or absence of copper for various times. FIG. 8A presentschanges in HIC Fraction #3 after 6 Hours, and FIG. 8B presents changesin HIC Fraction #3 after 18 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The invention provides methods of increasing the recovery ofactive recombinant proteins. In particular, the invention involvespromoting a desired conformation of a protein in preparations of arecombinant protein. Significantly, the invention provides gentlemethods of altering protein structure without necessitating the use ofharsh chaotrope treatments (such as, for example, strong denaturantssuch as SDS, guanidium hydrochloride or urea). Using the methods of theinvention on preparations of recombinant protein results in a higherpercentage, or higher relative fraction, of the recombinant protein inthe preparation with a desired conformation. A desired conformation fora recombinant protein is the three-dimensional structure of a proteinthat most closely resembles, and/or duplicates the function of, thenaturally occurring domain of that protein. Such gentle methods areparticularly advantageous when the recombinant protein is intended to beused in vivo as a drug or biologic.

[0016] Generally, when the recombinant protein contains a domain of areceptor protein, the desired conformation will have a higher bindingaffinity (and, consequently, a lower dissociation constant) for acognate ligand of the receptor. For example, the desired conformation ofa TNF-binding molecule will have a higher binding affinity and a lowerdissociation constant for TNF (e.g., TNF-alpha).

[0017] In addition, the desired conformation of a recombinant protein ispreferably more thermostable than an undesired conformation (whenmeasured in the same solution environment). Thermostability can bemeasured in any of a number of ways such as, for example, the meltingtemperature transition (Tm). The desired conformation of a recombinantprotein may or may not have a different arrangement of disulfide bonds,although preferably the conformation contains native disulfide bonds.The desired conformation of a recombinant protein may have othertertiary structure characteristics. For example, a desired conformationmay be a monomer, dimer, trimer, tetramer, or some other higher orderform of the protein. For the purposes of the invention, the“conformation” of a protein is its three-dimensional structure. Twodifferent structures of a polypeptide with the same primary amino acidsequence are “conformers” of each other when they have differentconformations corresponding to energy minima, and they differ from eachother only in the way their atoms are oriented in space. Conformers canbe interconverting (referring to the rotational freedom around bonds tothe exclusion of breaking bonds). Two different structures of apolypeptide with the same primary amino acid sequence are“configurational isomers” when they have different conformationscorresponding to energy minima, they differ from each other in the waytheir atoms are oriented in space, and they are non-interconvertiblewithout the breaking of a covalent bond. In the practice of theinvention, configurational isomers can be interconverted by, forexample, breaking and optionally reforming disulfidc bonds.

[0018] Thus, in one aspect, the invention entails contacting apreparation of the glycosylatcd recombinant protein that contains amixture of at least two configurational isomers of the recombinantprotein to a reduction/oxidation coupling reagent for a time sufficientto increase the relative proportion of the desired configurationalisomer and determining the relative proportion of the desiredconfigurational isomer in the mixture. In another aspect, the inventionentails contacting a preparation of a recombinant protein that has beenproduced by mammalian cells with a reduction/oxidation coupling reagent,at a pH of about 7 to about 11, and isolating a fraction of thepreparation of the recombinant protein with a desired conformation.Preferred recombinant proteins are glycosylated recombinant proteinssuch as, e.g., those produced by eukaryotic cells.

[0019] The invention can be used to treat just about any protein topromote a desired conformation. A protein is generally understood to bea polypeptide of at least about 10 amino acids, more preferably at leastabout 25 amino acids, even more preferably at least about 75 aminoacids, and most preferably at least about 100 amino acids. The methodsof the invention find particular use in treating proteins that have atleast about 3 cysteine residues, more preferably at least about 8cysteine residues, still more preferably at least about 15 cysteineresidues, yet even more preferably at least about 30, still even morepreferably at least about 50 to 150 cysteine residues.

[0020] Generally, the methods of the invention are useful for improvingproduction processes for recombinant proteins. Recombinant proteins areproteins produced by the process of genetic engineering. The term“genetic engineering” refers to any recombinant DNA or RNA method usedto create a host cell that expresses a gene at elevated levels, atlowered levels, and/or a mutant form of the gene. In other words, thecell has been transfected, transformed or transduced with a recombinantpolynucleotide molecule, and thereby altered so as to cause the cell toalter expression of a desired protein. Methods and vectors forgenetically engineering cells and/or cell lines to express a protein ofinterest are well known to those skilled in the art; for example,various techniques are illustrated in Current Protocols in MolecularBiology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, andquarterly updates) and Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Laboratory Press, 1989). Genetic engineeringtechniques include but are not limited to expression vectors, targetedhomologous recombination and gene activation (see, for example, U.S.Pat. No. 5,272,071 to Chappel) and trans activation by engineeredtranscription factors (see, for example, Segal el al., 1999, Proc. Natl.Acad. Sci. USA 96(6):2758-63).

[0021] The invention finds particular use in improving the production ofproteins that are glycosylated. Specifically, proteins that are secretedby fungal cell systems (e.g., yeast, filamentous fungi) and mammaliancell systems will be glycosylated. Preferably, the proteins are secretedby mammalian production cells adapted to grow in cell culture. Examplesof such cells commonly used in the industry are CHO, VERO, BHK, HeLa,CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (especiallymurine), PC12 and WI38 cells. Particularly preferred host cells areChinese hamster ovary (CHO) cells, which are widely used for theproduction of several complex recombinant proteins, e.g. cytokines,clotting factors, and antibodies (Brasel et al., 1996, Blood88:2004-2012; Kaufman et al., 1988, J.Biol Chem 263: 6352-6362; McKinnonet al., 1991, J Mol Endocrinol 6:231-239; Wood et al., 1990, J. Immunol145:3011-3016). The dihydrofolate reductase (DHFR)-deficient mutant cellline (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11and DG-44, are the CHO host cell lines of choice because the efficientDHFR selectable and amplifiable gene expression system allows high levelrecombinant protein expression in these cells (Kaufman R. J., 1990, MethEnzymol 185:527-566). In addition, these cells are easy to manipulate asadherent or suspension cultures and exhibit relatively good geneticstability. CHO cells and recombinant proteins expressed in them havebeen extensively characterized and have been approved for use inclinical manufacturing by regulatory agencies.

[0022] It has been found that the invention is a gentle and effectiveprocess for improving the production process for proteins that can adoptmultiple conformations and/or contain more than one domain. A “domain”is a contiguous region of the polypeptide chain that adopts a particulartertiary structure and/or has a particular activity that can belocalized in that region of the polypeptide chain. For example, onedomain of a protein can have binding affinity for one ligand, and onedomain of a protein can have binding affinity for another ligand. In athermostable sense, a domain can refer to a cooperative unfolding unitof a protein. Such proteins that contain more than one domain can befound naturally occurring as one protein or genetically engineered as afusion protein. In addition, domains of a polypeptide can havesubdomains.

[0023] In one aspect, the methods of the invention can be used onpreparations of recombinant proteins in which at least one domain of theprotein has a stable conformation, and at least one domain of theprotein has an unstable conformation. The terms “stable” and “unstable”are used as relative terms. The domain of the protein with a stableconformation will have, for example, a higher melting temperature (Tm)than the unstable domain of the protein when measured in the samesolution. A domain is stable compared to another domain when thedifference in the Tm is at least about 2° C., more preferably about 4°C., still more preferably about 7° C., yet more preferably about 10° C.,even more preferably about 15° C., still more preferably about 20° C.,even still more preferably about 25° C., and most preferably about 30°C., when measured in the same solution.

[0024] The invention is also generally applicable to proteins that havean Fc domain, and another domain (e.g., antibodies, and Fc fusionproteins). For example, in one of the non-limiting embodimentsillustrated below, TNFR:Fc, the Tm's for the Fc portion of the moleculeare at 69.1° C. and 83.4° C., while the Tm for the TNFR portion of themolecule range from 52.5° C. (in the more desired conformation) to a Tmof 49.7° C. (in the less desired conformation).

[0025] Particularly preferred proteins are protein-based drugs, alsoknown as biologics. Preferably, the proteins are expressed asextracellular products. Proteins that can be produced using the methodsof the invention include but are not limited to a flt3 ligand (asdescribed in WO 94/28391, which is incorporated by reference herein inits entirety), a CD40 ligand (as described in U.S. Pat. No. 6,087,329,which is incorporated by reference herein in its entirety),erythropoeitin, thrombopoeitin, calcitonin, Fas ligand, ligand forreceptor activator of NF-kappa B (RANKL), tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL, as described in WO97/01633, which is incorporated by reference herein in its entirety),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-CSF, asdescribed in Australian Patent No. 588819, which is incorporated byreference herein in its entirety), mast cell growth factor, stem cellgrowth factor, epidermal growth factor, RANTES, growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons, nerve growth factors, glucagon, interleukins 1 through 18,colony stimulating factors, lymphotoxin-β, tumor necrosis factor (TNF),leukemia inhibitory factor, oncostatin-M, and various ligands for cellsurface molecules ELK and Hek (such as the ligands for eph-relatedkinases or LERKS). Descriptions of proteins that can be purifiedaccording to the inventive methods may be found in, for example, HumanCytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwaland Gutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook (A. W. Thompson,ed., Academic Press, San Diego, Calif., 1991).

[0026] Preparations of the receptors, especially soluble forms of thereceptors, for any of the aforementioned proteins can also be improvedusing the inventive methods, including both forms of TNFR (referred toas p55 and p75), Interleukin-1 receptors types I and II (as described inEP 0 460 846, U.S. Pat. Nos. 4,968,607, and 5,767,064, which areincorporated by reference herein in their entirety), Interleukin-2receptor, Interleukin-4 receptor (as described in EP 0 367 566 and U.S.Pat. No. 5,856,296, which are incorporated by reference herein in theirentirety), Interleukin-15 receptor, lnterleukin-17 receptor,Interleukin-18 receptor, granulocyte-macrophage colony stimulatingfactor receptor, granulocyte colony stimulating factor receptor,receptors for oncostatin-M and leukemia inhibitory factor, receptoractivator of NF-kappa B (RANK, as described in U.S. Pat. No. 6,271,349,which is incorporated by reference herein in its entirety), receptorsfor TRAIL (including TRAIL receptors 1, 2, 3, and 4), and receptors thatcomprise death domains, such as Fas or Apoptosis-Inducing Receptor(AIR).

[0027] Other proteins whose production processes can be improved usingthe inventive methods include cluster of differentiation antigens(referred to as CD proteins), for example, those disclosed in LeukocyteTyping VI (Proceedings of the VIth International Workshop andConference; Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CDmolecules disclosed in subsequent workshops. Examples of such moleculesinclude CD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30ligand and CD40 ligand). Several of these are members of the TNFreceptor family, which also includes 41BB and OX40; the ligands areoften members of the TNF family (as are 41BB ligand and OX40 ligand);accordingly, members of the TNF and TNFR families can also be producedusing the present invention.

[0028] Proteins that are enzymatically active can also be preparedaccording to the instant invention. Examples includemetalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,and numerous other enzymes. Ligands for enzymatically active proteinscan also be expressed by applying the instant invention.

[0029] The inventive compositions and methods are also useful forpreparation of other types of recombinant proteins, includingimmunoglobulin molecules or portions thereof, and chimeric antibodies(e.g., an antibody having a human constant region coupled to a murineantigen binding region) or fragments thereof. Numerous techniques areknown by which DNA encoding immunoglobulin molecules can be manipulatedto yield DNAs capable of encoding recombinant proteins such as singlechain antibodies, antibodies with enhanced affinity, or otherantibody-based polypeptides (see, for example, Larrick et al., 1989,Biotechnology 7:934-938; Reichmann et al., 1988, Nature 332:323-327;Roberts et al., 1987, Nature 328:731-734; Verhoeyen et al., 1988,Science 239:1534-1536; Chaudhary et al., 1989, Nature 339:394-397).Preparations of fully human antibodies (such as are prepared usingtransgenic animals, and optionally further modified in vitro), as wellas humanized antibodies, can also be used in the invention. The termhumanized antibody also encompasses single chain antibodies. See, e.g.,Cabilly el al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. etal., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0519 596 A1. The method of the invention may also be used during thepreparation of conjugates comprising an antibody and a cytotoxic orluminescent substance. Such substances include: maytansine derivatives(such as DM1); enterotoxins (such as a Staphlyococcal enterotoxin);iodine isotopes (such as iodine-125); technium isotopes (such asTc-99m); cyanine fluorochromes (such as Cy5.5.18); andribosome-inactivating proteins (such as bouganin, gelonin, orsaporin-S6).

[0030] Examples of antibodies or antibody/cytotoxin orantibody/luminophore conjugates contemplated by the invention includethose that recognize any one or combination of the above-describedproteins and/or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14,CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86(B7.2), CD147, IL-1α, IL-1β, IL-4, IL-5, IL-8, IL-10, IL-2 receptor,IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunits,PDGF-β, VEGF, TGF, TGF-β2, TGF-β1, EGF receptor, VEGF receptor, C5complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen,LCG (which is a gene product that is expressed in association with lungcancer), HER-2, a tumor-associated glycoprotein TAG-72, the SK-1antigen, tumor-associated epitopes that are present in elevated levelsin the sera of patients with colon and/or pancreatic cancer,cancer-associated epitopes or proteins expressed on breast, colon,squamous cell, prostate, pancreatic, lung, and/or kidney cancer cellsand/or on melanoma, glioma, or neuroblastoma cells, the necrotic core ofa tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, the adhesionmolecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellularadhesion molecule-3 (ICAM-3), leukointegrin adhesin, the plateletglycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroidhormone, rNAPc2 (which is an inhibitor of factor VIIa-tissue factor),MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumornecrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, L-selectin, IFN-γ, Respiratory Syncitial Virus, humanimillimolarunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphlycoccus aureus.

[0031] Preparations of various fusion proteins can also be preparedusing the inventive methods. Examples of such fusion proteins includeproteins expressed as a fusion with a portion of an immunoglobulinmolecule, proteins expressed as fusion proteins with a zipper moiety,and novel polyfunctional proteins such as a fusion proteins of acytokine and a growth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO93/08207 and WO 96/40918 describe the preparation of various solubleoligomeric forms of a molecule referred to as CD40L, including animmunoglobulin fusion protein and a zipper fusion protein, respectively;the techniques discussed therein are applicable to other proteins. Anyof the above molecules can be expressed as a fusion protein includingbut not limited to the extracellular domain of a cellular receptormolecule, an enzyme, a hormone, a cytokine, a portion of animmunoglobulin molecule, a zipper domain, and an epitope.

[0032] The preparation of recombinant protein can be a cell culturesupernatant, cell extract, but is preferably a partially purifiedfraction from the same. By “partially purified” means that somefractionation procedure, or procedures, have been carried out, but thatmore polypeptide species (at least 10%) than the desired protein orprotein conformation is present. One of the advantages of the methods ofthe invention is that the preparation of recombinant protein can be at afairly high concentration. Preferred concentration ranges are 0.1 to 20mg/ml, more preferably from 0.5 to 15 mg/ml, and still more preferablyfrom 1 to 10 mg/ml.

[0033] The preparation of recombinant protein can be prepared initiallyby culturing recombinant host cells under culture conditions suitable toexpress the polypeptide. The polypeptide can also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a nucleotide sequence encoding thepolypeptide. The resulting expressed polypeptide can then be purified,or partially purified, from such culture or component (e.g., fromculture medium or cell extracts or bodily fluid) using known processes.Fractionation procedures can include but are not limited to one or moresteps of filtration, centrifugation, precipitation, phase separation,affinity purification, gel filtration, ion exchange chromatography,hydrophobic interaction chromatography (HIC; using such resins as phenylether, butyl ether, or propyl ether), HPLC, or some combination ofabove.

[0034] For example, the purification of the polypeptide can include anaffinity column containing agents which will bind to the polypeptide;one or more column steps over such affinity resins as concanavalinA-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one ormore steps involving elution; and/or immunoaffinity chromatography. Thepolypeptide can be expressed in a form that facilitates purification.For example, it may be expressed as a fusion polypeptide, such as thoseof maltose binding polypeptide (MBP), glutathione-S-transferase (GST) orthioredoxin (TRX). Kits for expression and purification of such fusionpolypeptides are commercially available from New England BioLab(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogcn,respectively. The polypeptide can be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. One such epitope (FLAG®) is commercially available from Kodak(New Haven, Conn.). It is also possible to utilize an affinity columncomprising a polypeptide-binding polypeptide, such as a monoclonalantibody to the recombinant protein, to affinity-purify expressedpolypeptides. Other types of affinity purification steps can be aProtein A or a Protein G column, which affinity agents bind to proteinsthat contain Fc domains. Polypeptides can be removed from an affinitycolumn using conventional techniques, e.g., in a high salt elutionbuffer and then dialyzed into a lower salt buffer for use or by changingpH or other components depending on the affinity matrix utilized, or canbe competitively removed using the naturally occurring substrate of theaffinity moiety. In one embodiment of the invention illustrated below,the preparation of recombinant protein has been partially purified overa Protein A affinity column.

[0035] Some or all of the foregoing purification steps, in variouscombinations, can also be employed to prepare an appropriate preparationof a recombinant protein for use in the methods of the invention, and/orto further purify the recombinant polypeptide after contacting thepreparation of the recombinant protein with a reduction/oxidationcoupling reagent. The polypeptide that is substantially free of othermammalian polypeptides is defined as an “isolated polypeptide”.

[0036] The polypeptide can also be produced by known conventionalchemical synthesis. Methods for constructing polypeptides by syntheticmeans are known to those skilled in the art. Thesynthetically-constructed polypeptide sequences can be glycosylated invitro.

[0037] The desired degree of final purity depends on the intended use ofthe polypeptide. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no polypeptide bandscorresponding to other polypeptides are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto the polypeptide can be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.Most preferably, the polypeptide of the invention is purified tosubstantial homogeneity, as indicated by a single polypeptide band uponanalysis by SDS-PAGE. The polypeptide band can be visualized by silverstaining, Coomassie blue staining, and/or (if the polypeptide isradiolabeled) by autoradiography.

[0038] By “contacting” is meant subjecting to, and/or exposing to, insolution. The protein or polypeptide can be contacted while also boundto a solid support (e.g., an affinity column or a chromatographymatrix). Preferably, the solution is buffered. In order to maximize theyield of protein with a desired conformation, the pH of the solution ischosen to protect the stability of the protein and to be optimal fordisulfide exchange. In the practice of the invention, the pH of thesolution is preferably not strongly acidic. Thus, preferred pH rangesare greater than pH 5, preferably about pH 6 to about pH 11, morepreferably from about pH 7 to about pH 10, and still more preferablyfrom about pH 7.6 to about pH 9.6. In one non-limiting embodiment of theinvention using TNFR:Fc that is illustrated below, the optimal pH wasfound to be about pH 8.6. However, the optimal pH for a particularembodiment of the invention can be easily determined experimentally bythose skilled in the art.

[0039] The reduction/oxidation coupling reagent is a source of reducingagents. Preferred reducing agents are free thiols. Thereduction/oxidation coupling reagent is preferably comprised of acompound from the group consisting of reduced and oxidized glutathione,dithiothreitol (DTT), 2-mercaptoethanol, dithionitrobenzoate, cysteineand cystine. For ease of use and economy, reduced glutathione and/orreduced cysteine can be used.

[0040] The reduction/oxidation coupling reagent is present at aconcentration sufficient to increase the relative proportion of thedesired conformation. The optimal concentration of thereduction/oxidation coupling reagent depends upon the concentration ofprotein and number of disulfide bonds in the protein. For example, ithas been found using a protein (TNFR:Fc) with 29 disulfide bonds at aconcentration of 2 mg/ml (approximately 14 microM protein or 400 microMdisulfide), a reduction/oxidation coupling reagent with 2 mM reducedthiols worked well to increase the relative proportion of the desiredconformation. This corresponds to a ratio of about 35 free thiols to 1disulfide bond. However, it was also found that ratios from 20 to 400free thiols per disulfide also worked. Of course, the amount of thiolused for a particular concentration can vary somewhat depending upon thereducing capacity of the thiol, and can be easily determined by one ofskill in the art.

[0041] Thus, generally, the concentration of free thiols from thereduction/oxidation coupling reagent can be from about 0.05 mM to about50 mM, more preferably about 0.1 mM to about 25 mM, and still morepreferably about 0.2 mM to about 20 mM.

[0042] In addition, the reduction/oxidation coupling reagent can containoxidized thiols at approximately higher, equal or lower concentrationsas the reduced thiol component. For example, the reduction/oxidationcoupling reagent can be a combination of reduced glutathione andoxidized glutathione. It has been found through actual working examples,that a ratio of reduced glutathione to oxidized glutathione of fromabout 1:1 to about 100:1 (reduced thiols:oxidized thiols) can functionequally well. Alternatively in another embodiment, thereduction/oxidation coupling reagent can be cysteine or a combination ofcysteine and cystine. Thus, when oxidized thiols are included in theinitial reduction/oxidation coupling reagent, the ratio of reducedthiols to oxidized thiols can in a preferred embodiment be from about1:10 to about 1000:1, more preferably about 1:1 to about 500:1, stillmore preferably about 5:1 to about 100:1, even more preferably about10:1.

[0043] Contacting the preparation of recombinant protein with areduction/oxidation coupling reagent is performed for a time sufficientto increase the relative proportion of the desired conformation. Anyrelative increase in proportion is desirable, but preferably at least10% of the protein with an undesired conformation is converted toprotein with the desired conformation. More preferably at least 20%,30%, 40%, 50%, 60%, 70% and even 80% of the protein is converted from anundesired to a desired conformation. Typical yields that have beenachieved with the methods of the invention range from 40 to 80%. If thecontacting step is performed on a partially or highly purifiedpreparation of recombinant protein, the contacting step can be performedfor as short as about 1 hour to about 4 hours, and as long as about 6hours to about 4 days. It has been found that a contacting step of about4 to about 16 hours or about 18 hours works well. The contacting stepcan also take place during another step, such as on a solid phase orduring filtering or any other step in purification.

[0044] The methods of the invention can be performed over a widetemperature range. For example, the methods of the invention have beensuccessfully carried out at temperatures from about 4° C. to about 37°C., however the best results were achieved at lower temperatures. Atypical temperature for contacting a partially or fully purifiedpreparation of the recombinant protein is about 4° C. to about 25° C.(ambient), but can also be performed at lower temperatures and at highertemperature.

[0045] The preparation of recombinant protein can be contacted with thereduction/oxidation coupling reagent in various volumes as appropriate.For example, the methods of the invention have been carried outsuccessfully at the analytical laboratory-scale (1-50 mL),preparative-scale (50 mL-10 L) and manufacturing-scale (10 L or more).Thus, the methods of the invention can be carried out on both small andlarge scale with reproducibility.

[0046] In preferred aspects, the contacting step is performed in theabsence of significant amounts of chaotropic agents such as, forexample, SDS, urea and guanidium HCl. Significant amounts of chaotropicagents are needed to observe perceptible unfolding. Generally, less than1 M chaotrope is present, more preferably less than 0.5 M, still morepreferably less than 0.1 M chaotrope. A solution is essentially free ofchaotrope (e.g., SDS, urea and guanidium HCl) when no chaotrope has beenpurposely added to the solution, and only trace levels (e.g., less than10 mM) may be present (e.g., from the vessel or as a cellularbyproduct).

[0047] Disulfide exchange can be quenched in any way known to those ofskill in the art. For example, the reduction/oxidation coupling reagentcan be removed or its concentration reduced through a purification step,and/or it can be chemically inactivated by, e.g., acidifying thesolution. Typically, when the reaction is quenched by acidification, thepH of the solution containing the reduction/oxidation coupling reagentwill be brought down below pH 7. Preferably, the pH is brought to belowpH 6. Generally, the pH is reduced to between about pH 2 and about pH 7.

[0048] Determining the conformation of a protein, and the relativeproportions of a conformation of a protein in a mixture, can be doneusing any of a variety of analytical and/or qualitative techniques. Ifthere is a difference in activity between the conformations of theprotein, determining the relative proportion of a conformation in themixture can be done by way of an activity assay (e.g., binding to aligand, enzymatic activity, biological activity, etc.). For example, inone of the non-limiting embodiments described below, at least twodifferent conformations of TNFR:Fc can be resolved by using asolid-phase TNF binding assay. The assay, essentially as described forIL-1R (Slack, et al., 1993, J. Biol. Chem. 268:2513-2524), candifferentiate between the relative proportions of various proteinconformations by changes in ligand-receptor binding association,dissociation or inhibition constants generated. Alternatively thebinding results can be expressed as activity units/mg of protein.

[0049] If the two conformations resolve differently duringchromatography, electrophoresis, filtering or other purificationtechnique, then the relative proportion of a conformation in the mixturecan be determined using such purification techniques. For example, inthe non-limiting embodiments described below, at least two differentconformations of TNFR:Fc could be resolved by way of hydrophobicinteraction chromatography. Further, since far-UV Circular Dichroism hasbeen used to estimate secondary structure composition of proteins(Perczel et al., 1991, Protein Engrg. 4:669-679), such a technique candetermine whether alternative conformations of a protein are present.Still another technique used to determine conformation is fluorescencespectroscopy which can be employed to ascertain complimentarydifferences in tertiary structure assignable to tryptophan and tyrosinefluorescence. Other techniques that can be used to determine differencesin conformation and, hence, the relative proportions of a conformation,are on-line SEC to measure aggregation status, differential scanningcalorimetry to measure melting transitions (Tm's) and componententhalpies, and chaotrope unfolding.

[0050] By the term “isolating” is meant physical separation of at leastone component in a mixture away from other components in a mixture.Isolating components or particular conformations of a protein can beachieved using any purification method that tends to separate suchcomponents. Accordingly, one can perform one or more chromatographysteps, including but not limited to HIC, hydroxyapatite chromatography,ion exchange chromatography, affinity, and SEC. Other purificationmethods are filtration (e.g., tangential flow filtration),electrophoretic techniques (e.g., electrophoresis, electroelution,isoelectric focusing), and phase separation (e.g., PEG-dextran phaseseparation), to name just a few. In addition, the fraction of thepreparation of recombinant protein that contains the protein in theundesired conformation can be treated again in the methods of theinvention, to further optimize the yields of protein with the desiredconformation.

[0051] For example, after treatment, protein samples can be prepared forhydrophobic interaction chromatography (HIC) by the following method. Anequal volume of 850 mM sodium citrate, 50 mM sodium phosphate, pH 6.5 isadded to the treated sample, and allowed to equilibrate to roomtemperature. After filtering (e.g., using a 0.22 □m filter), HICchromatography is performed on a Toyopearl® Butyl 650-M resin (TosohBiosep LLC, Montgomeryville, Pa.), at a flow rate of 150 cm/hr, and amass load of 2.1 mg•mL resin⁻¹. The column is prequilibrated with 3column volumes of 425 mM NaCitrate, 50 mM PO₄ pH 6.5, sample is loaded,and then washed through with 3 column volumes of 425 mM NaCitrate, 50 mMPO₄ pH 6.5. Elution can be performed with a gradient of 425 mMNaCitrate, 50 mM PO₄ pH 6.5 to O mM NaCitrate, 50 mM PO₄ pH 6.5 in atotal of 5 column volumes. Fractions can be collected during theelution. The column can be stripped with 3 column volumes of waterfollowed by 3 column volumes of 0.1 M NaOH. Using the methods of theinvention accordingly, one can thus obtain preparations of TNFR:Fc thatcontain more than 85%, more than 90%, and even more than 95% of theTNFR:Fc present in the preparation in the most active conformation(Fraction #2). Compositions, including pharmaceutical compositions, ofTNFR:Fc containing such proportions of Fraction #2 are therefore alsoprovided by the invention.

[0052] The invention also optionally encompasses further formulating theproteins. By the term “formulating” is meant that the proteins can bebuffer exchanged, sterilized, bulk-packaged and/or packaged for a finaluser. For purposes of the invention, the term “sterile bulk form” meansthat a formulation is free, or essentially free, of microbialcontamination (to such an extent as is acceptable for food and/or drugpurposes), and is of defined composition and concentration. The term“sterile unit dose form” means a form that is appropriate for thecustomer and/or patient administration or consumption. Such compositionscan comprise an effective amount of the protein, in combination withother components such as a physiologically acceptable diluent, carrier,and/or excipient. The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). Formulations suitablefor administration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. In addition, sterilebulk forms and sterile unit forms may contain a small concentration(approximately 1 microM to approximately 10 mM) of a reduction/oxidationcoupling reagent (e.g., glutathione, cysteine, etc.). The polypeptidescan be formulated according to known methods used to preparepharmaceutically useful compositions. They can be combined in admixture,either as the sole active material or with other known active materialssuitable for a given indication, with pharmaceutically acceptablediluents (e.g., saline, Tris-HCl, acetate, and phosphate bufferedsolutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens),emulsifiers, solubilizers, adjuvants and/or carriers. Suitableformulations for pharmaceutical compositions include those described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack PublishingCompany, Easton, Pa. In addition, such compositions can be complexedwith polyethylene glycol (PEG), metal ions, and/or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871;U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance, and are thus chosen according to the intended application, sothat the characteristics of the carrier will depend on the selectedroute of administration. Sustained-release forms suitable for useinclude, but are not limited to, polypeptides that are encapsulated in aslowly-dissolving biocompatible polymer (such as the alginatemicroparticles described in U.S. Pat. No. 6,036,978), admixed with sucha polymer (including topically applied hydrogels), and or encased in abiocompatible semi-permeable implant.

[0053] The invention having been described, the following examples areoffered by way of illustration, and not limitation. cl EXAMPLE 1

Biophysical Assessment of TNFR:Fc Fractions #2 and #3

[0054] TNFR:Fc elutes off a hydrophobic interaction column (HIC) asthree distinct peaks termed Fraction #1, Fraction #2 and Fraction #3(see FIG. 1). Fraction #2 is the desired fraction. Fraction #3 was ofparticular interest since it can comprise from 20 to 60% of the sampleand has been shown to exhibit low TNF binding activity and A375bioactivity in comparison with Fraction #2. Therefore, in the interestof understanding the differences between these two fractions andascertaining what factors contribute to the loss in activity forFraction #3 as it pertains to structure and conformation, biophysicalstudies were carried out. In this example, we analyzed Fraction #2 andFraction #3 using Circular Dichroism, Fluorescence, on-lineSEC/UV/LS/RI, and differential scanning calorimetry (DSC).

[0055] Materials and Methods

[0056] Materials: The starting material was TNFR:Fc in TMS buffer (10 mMTris, 4% mannitol, 1% sucrose). HIC eluted fractions of this materialwere isolated as Fractions #2 and #3 for experimental studies describedbelow.

[0057] Circular Dichroisim: Studies were carried out in the near(250-340 nm) and far-UV (190-250 nm) regions. The near-UV studies werecarried out to elucidate differences in tertiary structure while thefar-UV studies were used to characterize differences in secondarystructure.

[0058] The near-UV Circular Dichroism measurements were conducted in theTMS solutions with the following concentrations. Starting material wasdiluted to 6.25 mg/ml while the Fractions #2 and #3 were evaluated attheir existing concentrations of 9.4 and 5.4 mg/ml, respectively. ACircular Dichroism cell with a path length of 0.1 cm was used and scansconducted from 340 to 250 nm.

[0059] The far-UV Circular Dichroism measurements were performed withthe protein buffer exchanged into 10 mM sodium phosphate (pH 7.0) andsubsequently evaluated using a 0.1 cm path length cell scanned from 250to 190 nm. Secondary structure composition was evaluated using convexconstraint analysis (CCA) (Perczel et al., 1991, Protein Engrg.4:669-679).

[0060] Fluorescence Spectroscopy: Samples were examined after dilutionto approximately 50 microgram/ml using two different excitationwavelengths. Tyrosine and tryptophan fluorescence was examined with anexcitation of 270 nm while tryptophan fluorescence was exclusivelyevaluated using an excitation of 295 nm (Lakowicz, J. R. in “Principlesof Fluorescence Spectroscopy”, Plenum Press, 1983. New York, N.Y.,342-343). Fluorescence scans extended from 300 to 440 nm for 270 nmexcitation and from 310 to 440 nm for 295 nm excitation. Fourconsecutive scans were signal averaged for each spectrum. Normalizeddata were reported to evaluate differences in frequency arising from thesamples.

[0061] Online-SEC/UV/LS/RI: The molecular weights of eluting componentsusing size exclusion chromatography were ascertained using ultraviolet(UV @ 280 nm), light scattering (90°), and refractive index (RI)detection in series. This method has been well documented (see Arakawaet al., 1992, Anal. Biochem. 203:53-57 and Wen et al., 1996, Anal.Biochem. 240:155-166), and has an advantage of measuring thenonglycosylated molecular weights of proteins and peptides that areglycosylated. The SEC and UV data were collected using an Integral HPLCsystem (PerSeptive Biosystems, Inc.) with a BioSil-400-5 column (fromBioRad) using a flow rate of 1 ml/min. The elution buffer consisted of100 mM phosphate (pH 6.8) and 100 mM NaCl. A DAWN DSP multi-angle lightscattering detector and Optilab DSP refractometer were both purchasedfrom Wyatt Technology, Inc. Calibration standards to determineinstrumental constants included BSA dimer, BSA monomer and ovalbumin(FIG. 2).

[0062] Differential Scanning Calorimetry (DSC): Physical properties ofunfolding were measured using a MicroCal MC-2 DSC instrument in upscanmode. Samples were prepared by buffer exchanging into the same TMSbuffer at pH 7.4. Samples contained about 4 mg/ml protein and wereevaluated against the buffer (absent protein) as a reference. The scanrate was 67° C./hr spanning the temperature regime from 20° C. to 90° C.Collected scans were subsequently converted into concentrationnormalized scans to better compare enthalpic behavior of unfoldingtransitions while taking into account differences in concentration (datareported as kcal/mole).

[0063] Results

[0064] Circular Dicliroism. The near-UV Circular Dichroism measurementsexpressed in terms of mean residue ellipticity are shown in FIG. 2.Changes in a broad feature near 270 nm were evident between Fraction #2and #3 as shown by a greater proportion of negative ellipticity in thespectrum of Fraction #3 (indicated by the arrow in FIG. 2A). It wasnoted that the spectral behavior of the starting material closelymatches that of Fraction #2 but does exhibit a subtle negativedisplacement in the same region surrounding 270 nm. This result seemedconsistent as Fraction #3 made up a small part of the starting materialand so its contribution to the overall ellipticity in this region wasgreatly reduced but in the same displacement direction. Reproducibilityof the Fraction #3 spectrum confirmed the observed displacement of thissample to be real. With this in mind, and knowing that disulfides giverise to a broad negative elliptical feature in this region of theCircular Dichroism spectrum (see Kahn, P. C., 1978, Methods Enzymol.61:339-378 and Kosen et al., 1981, Biochemistry 20:5744-5754), thenear-UV Circular Dichroism spectrum was curve-fitted to estimate whatthe observed changes in this region mean in terms of tertiary structure.The results of the curve-fitted data are presented in FIG. 2B and showeda small red-shift (3 nm) and enhanced negative displacement consistentwith the contribution arising from a change in tertiary structureinvolving disulfides when comparing Fraction #3 with #2.

[0065] The far-UV Circular Dichroism has been used to estimate secondarystructure composition of proteins (Perczel et al., 1991, Protein Engrg.4:669-679). Secondary structure assignments using CCA were performed.Calculated spectra comprised of the sum of the secondary structureelements were compared with experimentally observed spectra andexhibited a good fit. The secondary structures of both fractions werecomparable within limits of experimental precision (within 10%).Therefore, this experiment did not distinguish any differences regardingsecondary structure for either of these two fractions.

[0066] Fluorescence Spectroscopy. Knowing that there were significantdifferences observed in the near-UV Circular Dichroism region,fluorescence spectroscopy was employed to ascertain complimentarydifferences in tertiary structure assignable to tryptophan and tyrosinefluorescence. Using two excitation wavelengths, it was possible todetermine that the spectra for all three cases considered (SM, Fraction#2 and #3) were super-imposable with fluorescence maxima near 338 nm.Since the three-dimensional structure of a given protein is responsiblefor emission maxima of native proteins, these results suggested that theaverage structure involving the intrinsic fluorophores, tryptophan andtyrosine was unperturbed.

[0067] On-line SEC/UV/LS/RI. The light scattering studies performedon-line with SEC yielded molecular weights of the main elution peak thatwere in agreement with the non-glycosylated polypeptide molecular weightof TNFR:Fc (e.g., 102 kD). Although there were clear differences in thecompositions of eluting species evaluated with this technique, whencomparing the elution profile of Fraction #3 with Fraction #2 (FIG. 3Aand B), the main peak comprising the majority component was measured tobe 102.5±1.6 kD (Retention Volume=8.4 mL) and 101.9±2.1 kD (RetentionVolume=8.3 mL), respectively. The precision was expressed as thestandard deviation of 23 slices through the elution peak bracketed bythe vertical dashed lines in FIG. 3. It was also noted that arespectable signal of the descending shoulder for Fraction #3 permitteddetermination of the polypeptide molecular weight to be 78.1±3.7 kD(this evaluation considered 8 slices surrounding the peak labeled at8.85 mL). As exhibited by the precision associated with the molecularweight determination of this component, this peak exhibited greaterheterogeneity and as a result was suspect of greater polydispersion thanthe main peak. Fraction #3 also contained a significant amount of highmolecular weight species consistent with the elution volume of apredominantly dimeric form of TNFR:Fc (near 7.5). Hence, it wasdetermined that Fraction #3 is comprised of several species includingaggregates and fragmented portions of the molecule.

[0068] Differential Scanning Calorimetry. DSC measurements carried outon the two fractions yielded significant differences in the unfolding ofthe TNFR moiety of the TNFR:Fc molecule (FIG. 4). As shown more clearlyin the baseline corrected data (FIG. 4B), there is a 2.8° C. shift tolower temperature in the melting transition (Tm) when comparing a Tm of52.5° C. (Fraction #2) with a Tm of 49.7° C. (Fraction #3). Thetransition is slightly broader for Fraction #3 with a half-width at halfthe transition maximum of 8° C. in comparison with Fraction #2 having ahalf-width of 6.5° C. This low temperature transition has beenidentified from thermal unfolding experiments of TNFR:Fc monomer to bedue the TNFR domain of the molecule. Thermal transitions at 69.1° C. and83.4° C. have been assigned to the Fc portion of the molecule. Theselatter two unfolding transitions align well and are comparable in termsof Tm's and component enthalpies.

[0069] Discussion

[0070] Among the methods tested, differences were observed in thenear-UV Circular Dichroism and DSC measurements. Differential scanningcalorimetry data support a loosening of structure that is assignable tothe receptor moiety of the molecule with little change observed in theregion of the Fc. The near-UV Circular Dichroism results suggested thatdisulfides are involved with tertiary structural changes associated withFraction #3. These changes may arise as a consequence of burieddisulfides gaining more exposure to the solvent and account for anincrease in hydrophobicity as suggested by the small increase inretention time observed in the HIC elution of Fraction #3. It isinteresting that there are no discernible differences found in thefluorescence data that would indicate such a change in conformationalstructure. If one considers the primary structure of TNFR:Fc in terms ofthe distribution of tyrosines (Y) and tryptophans (W), it becomesapparent that the region extending from the C-terminal portion ofresidue 104 of the TNFR domain to residue 296 of the N-terminal portionof the Fc (comprising 40% of the linear sequence of TNFR:Fc) is devoidof these intrinsic fluorophores. Therefore, one possible explanationconsistent with the data might be that tertiary structure remote fromthe Fc hinge region is relatively unchanged while that from aboutresidue C115 to C281 may be somewhat altered conformationally. Thisregion of the molecule comprises 10 possible cysteines that may beaffected with supposedly little consequence of structural changeaffecting local structure of tyrosines and tryptophans. It is noted thatit is currently unknown as to how this molecule is folded and it wouldseem plausible that the cysteines that make up disulfides that are moreremote from any given tryptophan or tyrosine residue would be logicalsuspects for tertiary structural changes that produce the observednear-UV Circular Dichroism results but exhibit little impact on thevicinal structure involving tyrosines and tryptophans. This idea doesnot preclude the possibility that there is some unusual change instructure within one or both of the TNFR arms that does not invoke anappreciable change in the net effect of fluorescence arising fromtyrosines and tryptophans. The fact that the fluorescence data (which isinsensitive to disulfides) show no change and the near-UV (that issensitive to disulfides, tyrosines, and tryptophans) exhibits a smallnegative displacement consistent with disulfide structural modificationdoes imply that disulfides play a role in the difference betweenFractions #2 and #3.

[0071] In summarizing the remaining data generated concerning Fraction#3, aspects related to molecular weight and secondary structure werefound to be indistinguishable from Fraction #2.

EXAMPLE 2 Disulfide Exchange Experiments on TNFR:Fc Fraction #3 withGlutathione

[0072] This experiment was designed to assess a variety of treatments todrive TNRF:Fc Fraction #3 into the conformation of Fraction #2 in aprocess amenable to large-scale production runs.

[0073] Materials and Methods

[0074] Materials. The starting material was TNFR:Fc as a Protein Aelute, a pure HIC elute of Fraction #3, and a 50:50 mixture of HICelutes Fraction #2 and Fraction #3. Buffers were 0.1 M citrate or 0.1 MTris/glycine at pH 7.6, pH 8.6 or pH 9.6. Protein concentration of theTNFR:Fc was from 0.2 to 4.5 mg/mL. A redox coupling system of reducedglutathione and glutathione (GSH/GSSG at a ratio of 10:1) was added at0.1 to 5 mM GSH. Incubation temperature was varied at 4 degrees, 22degrees or 31 degrees Centigrade.

[0075] Methods. Disulfide exchange was quenched by acidification of thesample to pH 6 with 1 M acetic acid. Treated preparations of recombinantprotein were characterized by analytical HIC, SEC (retention time,aggregate concentration) and solid-phase TNF binding assay to determinethe percentage and yield of Fraction #2.

[0076] Results and Discussion

[0077] Treatment efficiency as a function of pH and GSH concentration.Significant % of the protein in Fraction #3 (at least 10%) was convertedFraction #2 when treatment was performed at both 0.1 mM GSH/pH 7.6 and0.1 mM GSH/pH 8.6. However, efficiency was greatly improved (from 45% toalmost 70%) when treatment was performed at 0.1 mM GSH/pH 9.6; 1 mMGSH/pH 7.6; 1 mM GSH/pH 8.6; and 1 mM GSH/pH 9.6. Thus, althoughtreatment efficiency is sensitive to pH and free thiol concentration, itcan be effectively performed over a wide range of these variables.

[0078] Temperature effects. Fraction #3 was treated at three differenttemperatures, 4° C., 22° C. and 31° C. The GSH concentration was held at1 mM, and pH 8.6. After 16 hours, the treatment groups all exhibitedsignificant conversion of Fraction #3 into Fraction #2, but conversionseemed slightly more efficient at the two lower temperatures.

[0079] Clone effects. Six different cell line clones, all producingTNFR:Fc, were tested in a standardized protocol based upon the aboveresults. Specifically, a Protein A elution containing 0.4 to 0.7 mg/mLof TNFR:Fc (at about pH 4) was adjusted to pH 8.6 using 1 M Tris/glycine(final concentration 0.1 M Tris/glycine). These solutions were adjustedto 1 mM EDTA and 2.5 mM GSH/0.25 mM GSSG and incubated at roomtemperature for about 16 hours. Disulfide exchange was quenched byacidification as described above.

[0080] Each of six different clones all showed improvement in productionand yield of Fraction #2. The reduction of HIC Fraction #3 by treatmentin the various clones was 64%, 72%, 77%, 78%, 78% and 83%. The increasein HIC Fraction #2 in the same clones was 37%, 64%, 78%, 70%, 44% and54%, respectively. Percent increase in HIC Fraction #2 was wellcorrelated with the % increase in Binding Units, as shown in FIG. 5.Thus, the methods appeared generally applicable across all clonestested.

[0081] Binding assays. Three different preparations of TNFR:Fc wereassayed in a solid-phase TNF binding assay. Samples 11-6 and 12 wereeluants from a Protein A column. Sample 8085-47 was also eluted from aProtein A column, and then subjected to an additional HIC purificationstep; this sample contained exclusively Fraction #3. Samples wereexamined in the binding assay before and after disulfide exchange asdescribed above. The results presented below in Table 1 show an increasein ligand binding activity after treatment of all samples withglutathione. TABLE 1 TNF binding activity of TNFR:Fc before and afterdisulfide exchange Pre-exchange Post-exchange % Sample (activity/mg ofprotein) Change 11-6 4.16 × 10⁷ 5.73 × 10⁷ 27% 12 4.36 × 10⁷ 6.13 × 10⁷29% 8085-47 1.90 × 10⁷ 6.75 × 10⁷ 72%

EXAMPLE 3 Disulfide Exchange Experiments on TNFR:Fc Treated withL-Cysteine

[0082] This experiment was designed to assess cysteine/cystine asreduction/oxidation coupling reagents for TNFR:Fc. The procedure allowsassessment of change of HIC Fraction #3 into the conformation ofFraction #2 in a process amenable to large-scale production runs. Theprocedure can be performed on a purified Fraction #3, a mixture ofFractions #2 and #3, and/or following other separation techniques suchas Protein A chromatography, with similar results.

[0083] Materials and Methods

[0084] The starting material was TNFR:Fc as a pure HIC elute of Fraction#3 or as a Protein A-eluted TNFR:Fc containing both Fraction #2 and #3.Buffers were 0.1 M citrate or 0.2 M Tris at pH 8.5. Proteinconcentration of the TNFR:Fc was 2.5 to 3 mg/mL.

[0085] A redox coupling system of L-cysteine (varying from 0 to 50 mM)was utilized. The procedure was also assessed +/−L-cystine (0.025 to 0.5mM) and +/−1 mM EDTA. Incubation temperature was assessed at 4, 15, and22 degrees Centigrade for 6, 18, and 48 hours. Disulfide exchange wasquenched by acidification of the sample to pH 7 with NaH₂PO₄ or 0.85 Mcitrate. Treated preparations of recombinant protein were characterizedby analytical HIC and SEC (retention time, aggregate concentration) todetermine the percentage and yield of Fraction #2 and Fraction #3,cysteinylation and free sulfhydral assays.

[0086] Results and Discussion

[0087] Treatment efficiency as a function of L-cysteine concentration(0-5 mM). A significant percentage of the TNFR:Fc protein in HICFraction #3 (average 10%) was converted to Fraction #2 when treatmentwas performed with 0.25 mM L-cysteine in the absence of L-cystine orEDTA in four replicate samples (FIG. 6). However, efficiency was greatlyimproved (from 45% to almost 70%) when treatment was performed at 1 mML-cysteine or 5 mM L-cysteine (FIG. 6). The effect of cystine in thesereaction conditions varied with EDTA presence (see below). For a givencell culture batch (samples from four different cell culture batcheswere treated), the treatment process was reproducible.

[0088] Treatment efficiency as a function of higher L-cysteineconcentration (5-50 mM). Higher concentrations of L-cysteine (5, 15 and50 mM L-cysteine) used to treat TNFR:Fc resulted in a decrease in HICFraction #3 from the starting material in each case, but 5 mM L-cysteinewas most effective in promoting the accumulation of Fraction #2 (FIG.7). It is estimated that higher concentrations of L-cysteine eithersignificantly reduced the sulfhydryl moieties in the molecule orrequired too long to re-oxidize.

[0089] Treatment efficiency as a function of additional L-cysteinefeeding. In order to attempt to increase disulfide exchange efficiency,TNFR:Fc was treated with 5 mM L-cysteine and incubated at 4 degreesCentigrade for 18 hours. Additional L-cysteine (0-5 mM) was then added,and the samples incubated at 4 degrees Centigrade for two additionaldays. Under these conditions, no significant effect on the ratio of HICFraction #3 to Fraction #2 was noted by additional L-cysteine feeding.

[0090] Effect of EDTA, cystine and L-cysteine. The effect of cystine(0-0.4mM) in combination with L-cystcine (5 mM) on TNFR:Fc was assessedin the presence or absence of 1 mM EDTA. Optimal results in the presenceof 1 mM EDTA occurred with concentrations of cystine in the range of0.1-0.2 mM.

[0091] Copper, temperature and time effects. TNFR:Fc was treated at with5 mM L-cysteine at 4 degrees and 22 degrees Centigrade for either 6 or18 hours. Completion of treatment of TNFR:Fc was assayed by copperaddition followed by HIC. After 6 hours of incubation, disulfideexchange is more complete at 4 degrees than 22 degrees, and treatment isclearly more complete after 18 hours at 4 degrees Centigrade (FIGS. 8Aand 8B).

[0092] Comparison of analytical- versus preparative-scale L-cysteinetreatment efficiency. Based upon the treatment conditions optimized atsmall scale, TNFR:Fc (2.5 mg/mL in 0.2 M Tris, pH 8.5) in either 3 mL or20 L quantities were treated with 5 mM L-cysteine (in the absence ofcystine or EDTA), incubated at 4 degrees Centigrade for 18 hours,diluted with and equal volume of 850 mM sodium citrate, 50 mM sodiumphosphate, pH 6.5 to quench the treatment, and chromatographed on HIC.Control samples of Preparative and Analytical scale TNFR:Fc had 63% and68% Fraction #3, respectively. After treatment with the aboveconditions, Fraction #3 was reduced to 28% in both Preparative andAnalytical scales. Therefore the treatment efficiency was 56% and 59%for the Preparative and Analytical samples, respectively (Table 2). Thisexperiment demonstrates that the process is amenable to larger scaletreatment. TABLE 2 Analytical vs. Preparative Scale Disulfide ExchangeProcedure PREPARATIVE ANALYTICAL Fraction #2 Fraction #3 Fraction #2Fraction #3 Control 37% 63% 32% 68% Exchange 72% 28% 72% 28% Exchange“efficiency” 56% 59%

[0093] Thus, although treatment redox efficiency is affected by freethiol concentration, temperature and time, it can be effectivelyoptimized and performed over a wide range of variables. The treatmentprotocols can also be performed on both small and large scale withreproducibility.

[0094] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A method comprising: contacting a preparation ofa recombinant protein that has been produced by mammalian cells with areduction/oxidation coupling reagent, at a pH of about 7 to about 11,and isolating a fraction of the preparation of the recombinant proteinwith a desired conformation.
 2. The method of claim 1 wherein therecombinant protein contains at least two domains.
 3. The method ofclaim 2 wherein at least one domain of the protein has a stableconformation, and at least one domain of the protein has an unstableconformation.
 4. The method of claim 1 wherein the recombinant proteincomprises an extracellular domain of a receptor.
 5. The method of claim1 wherein the recombinant protein is a soluble form of a TNF-receptor.6. The method of claim 1 wherein the recombinant protein is a Fc fusionprotein.
 7. The method of claim 6 wherein the preparation of therecombinant protein has been purified from a Protein A or Protein Gcolumn.
 8. The method of claim 1 wherein the recombinant protein isselected from the group consisting of a soluble IL-4 receptor, a solubleIL-1 type II receptor, a soluble Flt3 ligand, a soluble CD40 ligand,CD39, CD30, CD27, a TEK/Ork, IL-15, a soluble IL-15 receptor, Ox 40,GM-CSF, RANKL, RANK, TRAIL, a soluble TRAIL receptor, tissue plasminogenactivator, Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I,an IL-2 receptor, an IL-2 antagonist, alpha-1 antitrypsin, calcitonin,growth hormone, insulin, insulinotropin, an insulin-like growth factor,parathyroid hormone, an interferon, superoxide dismutase, glucagon, anerythropoeitin, an antibody, glucocerebrosidase, an Fc-fusion protein, aglobin, a nerve growth factor, an interleukin, and a colony stimulatingfactor.
 9. The method of claim 1 wherein the pH is from about 7 to about10.
 10. The method of claim 9 wherein the pH is about 7.6 to about 9.6.11. The method of claim 10, wherein the pH is about 8.6.
 12. The methodof claim 1 wherein the reduction/oxidation coupling reagent comprisesglutathione.
 13. The method of claim 12 wherein the ratio of reducedglutathione to oxidized glutathione is about 1:1 to about 100:1.
 14. Themethod of claim 1 wherein the reduction/oxidation coupling reagentcomprises cysteine.
 15. The method of claim 1 wherein the contactingstep is performed for about 4 to about 16 hours.
 16. The method of claim1 wherein the contacting step is performed at about 25° C.
 17. Themethod of claim 1 wherein the contacting step is performed at about 4°C.
 18. The method of claim 1 wherein the contacting step is quenched byacidification.
 19. The method of claim 1 wherein the isolating stepcomprises one or more chromatography steps.
 20. The method of claim 1wherein the protein concentration is from about 0.5 to about 10 mg/ml.21. The method of claim 1 wherein the ratio of reducing thiols in thereduction/oxidation coupling reagent to disulfide bonds in the proteinis about 320:1 to about 64,000:1 (reducing thiols: disulfide bond). 22.The method of claim 1 further comprising formulating the fraction of thepreparation of the recombinant protein with the desired conformation ina sterile bulk form.
 23. The method of claim 1 further comprisingformulating the fraction of the preparation of the recombinant proteinwith the desired conformation in a sterile unit dose form.
 24. Themethod of claim 4 wherein the desired conformation has a higher bindingaffinity for a cognate ligand of the receptor.
 25. The method of claim 5wherein the desired conformation has a higher binding affinity for TNF.26. The method of claim 25 wherein the TNF is TNF-alpha.
 27. A method ofpromoting a desired conformation of a glycosylated recombinant protein,the method comprising contacting a preparation of the glycosylatedrecombinant protein that contains a mixture of at least twoconfigurational isomers of the glycosylated recombinant protein with areduction/oxidation coupling reagent for a time sufficient to increasethe relative proportion of the desired configurational isomer anddetermining the relative proportion of the desired configurationalisomer in the mixture.
 28. The method of claim 27 wherein theglycosylated recombinant protein contains at least two domains.
 29. Themethod of claim 28 wherein at least one domain of the glycosylatedrecombinant protein has a stable conformation, and at least one domainof the glycosylated recombinant protein has an unstable conformation.30. The method of claim 27 wherein the glycosylated recombinant proteincomprises an extracellular domain of a receptor.
 31. The method of claim27 wherein the glycosylated recombinant protein is a soluble form of aTNF-receptor.
 32. The method of claim 27 wherein the glycosylatedrecombinant protein is a Fc fusion protein.
 33. The method of claim 32wherein the preparation of the glycosylated recombinant protein has beenpurified from a Protein A or Protein G column.
 34. The method of claim27 wherein the glycosylated recombinant protein is selected from thegroup consisting of a soluble IL-4 receptor, a soluble IL-1 type IIreceptor, a soluble Flt3 ligand, a soluble CD40 ligand, CD39, CD30,CD27, a TEK/Ork, IL-15, a soluble IL-15 receptor, Ox 40, GM-CSF, RANKL,RANK, TRAIL, a soluble TRAIL receptor, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, an IL-2receptor, an IL-2 antagonist, alpha-1 antitrypsin, calcitonin, growthhormone, insulin, insulinotropin, an insulin-like growth factor,parathyroid hormone, an interferon, superoxide dismutase, glucagon, anerythropoeitin, an antibody, glucocerebrosidase, an Fc-fusion protein, aglobin, a nerve growth factor, an interleukin, and a colony stimulatingfactor.
 35. The method of claim 27 wherein the pH is from about 7 toabout
 10. 36. The method of claim 35 wherein the pH is about 8.6. 37.The method of claim 27 wherein the reduction/oxidation coupling reagentis selected from the group consisting of glutathione, cysteine, DTT(dithiothreitol), 2-mercaptoethanol and dithionitrobenzoate.
 38. Themethod of claim 37 wherein the reduction/oxidation coupling reagentcomprises reduced glutathione.
 39. The method of claim 38 wherein thereduced glutathione is at a concentration of about 1 mM to about 10 mM.40. The method of claim 37 wherein the reduction/oxidation couplingreagent comprises reduced cysteine.
 41. The method of claim 37 whereinthe ratio of reducing thiols in the reduction/oxidation coupling reagentto disulfide bonds in the protein is about 320:1 to about 64,000:1(reducing thiols: disulfide bond).
 42. The method of claim 27 whereinthe protein concentration is from about 0.5 to about 10 mg/ml.
 43. Themethod of claim 27 wherein the contacting step is performed for about 4to about 16 hours.
 44. The method of claim 27 wherein the contactingstep is performed at about 25° C.
 45. The method of claim 27 wherein thecontacting step is performed at about 4° C.
 46. The method of claim 27wherein the contacting step is quenched by acidification.
 47. The methodof claim 27 wherein the determining step comprises one or morechromatography steps.
 48. The method of claim 27 wherein the determiningstep comprises a binding reaction.
 49. The method of claim 27 comprisingisolating a fraction of the preparation of the glycosylated recombinantprotein with the desired configurational isomer.
 50. The method of claim49 comprising formulating the desired configurational isomer in asterile unit dose form.
 51. The method of claim 30 wherein the desiredconfigurational isomer has a higher binding affinity for a cognateligand of the receptor.
 52. The method of claim 31 wherein the desiredconfigurational isomer has a higher binding affinity for TNF.
 53. Themethod of claim 52 wherein the TNF is TNF-alpha.
 54. A method comprisingformulating into sterile unit dose form a recombinant protein that hasbeen produced by mammalian cells, contacted with a reduction/oxidationcoupling reagent, and isolated from the fraction of the protein with anundesired conformation.
 55. A pharmaceutical composition of a TNFR:Fcproduced by the method of claim 54.